The role of microglia and infiltrating monocytes in experimental autoimmune encephalomyelitis (EAE) pathogenesis has been controversial. To gain insight into their respective roles, we developed a method for differentiating between microglia and infiltrating monocytes in the CNS using CD44. We used this system to monitor changes in cell number, activation status, and gene expression by RNA sequencing (RNA-Seq) over the course of disease. This in vivo characterization and RNA-Seq dataset improves our understanding of myeloid cell biology in the brain under inflammatory conditions and may lead to strategies to identify therapies for inflammatory pathways active in neuroinflammatory diseases. Pooled samples from 10 mice were analyzed for both microglia and monocytes at distinct timepoints post EAE inducation. Peritoneal macrophages were isolated and analyzed for five samples, and also in a pool.